Mitochondrial dysfunction causes neurodegenerative diseases. Energy deficiency concomitant with increased free radical production is responsible for cell death in many of the early-onset mitochondrial disorders. It is now increasingly appreciated that mitochondrial dysfunction also plays an important role in adult- and late-onset neurodegenerative diseases. However, a role of bioenergetic deficit as the primary cause of these diseases is less compelling. The mitochondrial processes affected in the degenerative diseases are often not directly involved in oxidative phosphorylation, which include protein quality control, organellar dynamics, mitophagy and stress signaling among others. Protein quality control on the inner membrane of mitochondria (IMM) is dependent on a group of highly conserved proteases, including the m-AAA protease, the presenilin-associated rhomboid-like protease (PARL), the HTRA2 protease (PARK13) and the IMMP2 processing peptidase. Defects in these proteases cause neurodegenerative diseases such as spinocerebellar ataxia, spastic ataxia-neuropathy syndrome, spastic paraplegia and Parkinson's disease. The pathogenic mechanisms underlying these diseases are unclear. Two pertinent questions are still outstanding: (1) is protein misfolding and proteostatic stress on the IMM the primary cause of neurodegeneration? and (2) if so, how does it induce neuronal cell death? The multifunctionality of the protein quality control proteases prevented their utility for testing potential non-bioenergetic roles of these enzymes in neuronal survival. To overcome this obstacle, we recently generated a mouse model that expresses a misfolded and pathogenic variant of the adenine nucleotide translocase 1 (Ant1) on the IMM. Our preliminary studies showed that the misfolded Ant1 accumulates specifically in the central nervous system, which induces an ascending paralytic phenotype. We also showed that introduction of similar mutations in yeast induce cell death by a novel mechanism that we named mitochondrial Precursor Over-accumulation Stress (mPOS). mPOS is characterized by the toxic accumulation of mitochondrial precursor proteins in the cytosol. Thus, clinically relevant mitochondrial damage is sufficient to induce proteostatic stress in the cytosol. In this grant application, we will test the hypothesis that mPOS plays a role in neurodegeneration. In Specific Aim 1, we will test the hypothesis that proteostatic stress on the IMM induces selective cell death in the central nervous system particularly in the lower regions of the spinal cord. In Specific Aim 2, we will test the hypothesis that proteostatic stress on the IMM induces mPOS, which contributes to neurodegeneration. Success of the project will help our understanding of many neurodegenerative diseases induced by proteostatic stress on the IMM. It may also have implications for motor neuron diseases (e.g., ALS and hereditary spastic paraplegia), in which the contribution of mitochondrial dysfunction to the pathogenesis is poorly defined.